A variety of studies conducted at the ultrastructural and biochemical levels have attempted to elucidate the basic mechanisms of cellular aging by comparing the properties of human diploid cells at early passage in culture to those of late passage cultures. It has become evident, however, that two physiologically distinct subpopulations emerge in senescent cultures of human fibroblasts. The postmitotic cells, comprising one subpopulation, have larger nuclei and a larger modal cell volume than do the actively proliferating fibroblasts which comprise the other subpopulations. Data obtained on whole senescent cultures are therefore representative of a mixed population and do not characterize individually either the actively proliferating or the postmitotic fibroblasts. The objective of this proposed research is to characterize these two subpopulations by measuring the quantitative changes that accompany senescence in human diploid fibroblasts in: 1) the volume density (cubed microns) of nuclei, nucleoli, mitochondria, smooth and rough endoplasmic reticulum, golgi, lysosomes, secretory granules, pigment granules and lipid droplets, 2) the surface density (sq. microns) of nuclear envelope, smooth and rough endoplasmic reticulum and golgi, 3) the line density (microns/cell) of microtubules and microfilaments and 4) the numerical density (no./cell) of ribosomes. This will be accomplished in experiments where sterological analysis will be conducted on autoradiograms of early, middle and late passage cultures previously exposed to 3H-TdR. As a measure of changes in the intracellular environment, electron microprobe analysis will be used to determine (mmole/kg dry wt.) the cytoplasmic and intranuclear concentrations of Na, K, Cl, Mg, P and S during in vitro senescence and under conditions where the proliferative capacity of each analyzed cell will be tested. Quantitative changes both in organelle systems and in intracellular electrolyte concentrations can be directly associated with proliferating and postmitotic fibroblasts during in vitro aging. These determinations will be made in three cell lines from donors of varying age and in cultures experimentally induced to extend their in vitro lifespan with hydrocortisone treatment. The data obtained from this study will better enable us to understand the transition from the proliferating to the postmitotic state during cellular aging.